The calcium/ calcineurin/ NFAT pathway is essential for the adaptive immune response, a point underscored by the clinical efficacy of the calcineurin inhibitors cyclosporin A (CsA) and FK506. NFAT also plays an important role in the development and function of many other organs and cell types - neurons, heart, skeletal and smooth muscle, bone, skin, pancreas, and the vasculature. Surprisingly, however, there has not yet been a detailed examination, at a genome-wide level, of how NFAT transcription factors bind cis-regulatory DNA elements and change the transcriptional profiles of cells. In this application, we address this point for CD4+ Th1 and CD8+ cytolytic T cells (CTL). A striking feature of NFAT proteins is their ability to form tight complexes with unrelated transcription factors such as AP-1 (Fos-Jun) on composite elements in DNA. We have shown that NFAT in the absence of its partner AP-1 induces a negative regulatory programme of gene expression (T cell 'anergy' or 'exhaustion') that is distinct from the programme of T cell activation induced by NFAT: AP-1 complexes. Indeed, our preliminary data suggest that in the absence of AP-1, NFAT potentially functions as a 'master regulator' of CD8+ T cell exhaustion, a hypothesis that will be tested here. In Aim 1 of the application, we will examine the binding of the three immune-related NFAT proteins - NFAT1, NFAT2 and NFAT4 - to regulatory regions in genomic DNA of Th1 cells and CTL, under conditions of cooperation or lack of cooperation with AP-1. We will perform chromatin immunoprecipitation (ChIP) followed by next-generation sequencing (ChIP-seq), and a variant procedure known as ChIP-exo that more closely defines the binding sites for these transcription factors in DNA. We will also define the gene expression patterns regulated by these wild type and mutant NFAT proteins, by performing RNA-sequencing (RNA-seq) for steady-state or 'nascent' (chromatin-associated) RNA-sequencing on cells expressing the proteins. In Aim 2, we will repeat these experiments in human T cells. In Aim 3, we will implement a novel high-throughput screen to identify compounds that block the NFAT: AP-1 interaction without affecting the binding of NFAT to DNA or the formation of NFAT complexes with other partners such as FOXP3. In Aim 4, we will relate the patterns of gene expression defined in vitro in Aims 1 and 2 to the patterns obtained during immune responses in vivo, and ask whether T cells expressing mutant NFAT proteins that cannot cooperate with AP-1 induce a more profound phenotype of T cell anergy/ exhaustion during viral infections. From a clinical perspective, our project is very relevant to cancer immunotherapy and the treatment of viral infections, since CD8+ T cell exhaustion limits T cell responses in tumour-infiltrating CTL and during chronic viral infections.